Recently it has been established that a family of enzymes called Nitric Oxide Synthase (xe2x80x9cNOSxe2x80x9d) form nitric oxide from L-arginine, and the nitric oxide produced is responsible for the endothelium dependent relaxation and activation of soluble guanylate cyclase, neurotransmission in the central and peripheral nervous systems, and activated macrophage cytotoxicity.
Nitric Oxide Synthase, occurs in many distinct isoforms which include a constitutive form (cNOS) and an inducible form (iNOS). The constitutive form is present in normal endothelial cells, neurons and some other tissues. Formation of nitric oxide by the constitutive form in endothelial cells is thought to play an important role in normal blood pressure regulation, prevention of endothelial dysfunction such as hyperlipodemia, arteriosclerosis, thrombosis, and restenosis. The inducible form of nitric oxide synthase has been found to be present in activated macrophages and is induced in vascular smooth muscle cells, for example, by various cytokines and/or microbial products.
The conversion of precursor substrates of EDNO such as L-arginine into nitric oxide is enzymatically catalyzed by NOS and the resulting by-product of the conversion of L-arginine is L-citrulline. Although it was initially described in endothelium, NOS activity has now been described in many cell types. Brain, endothelium, and macrophage isoforms appear to be products of a variety of genes that have approximately 50% amino acid identity. NOS in brain and in endothelium have very similar properties, the major differences being that brain NOS is cytosolic and the endothelial enzyme is mainly a membrane-associated protein.
Functionally, the constitutive form of Nitric Oxide Synthase (xe2x80x9ccNOSxe2x80x9d), which is the predominant synthase present in brain and endothelium, may be active under basal conditions and can be further stimulated by increases in intracellular calcium that occur in response to receptor-mediated agonists or calcium ionophores. cNOS appears to be the xe2x80x9cphysiologicalxe2x80x9d form of the enzyme and plays a role in a diverse group of biologic processes. In vitro studies suggest that the activity of nitric oxide synthase can be regulated in a negative feedback manner by nitric oxide itself.
In contrast to the cNOS, the inducible, calcium-independent form, iNOS was initially only described in macrophages. It is now known that induction of nitric oxide synthase can occur in response to appropriate stimuli in many other cell types. This includes both cells that normally do not express a constitutive form of nitric oxide synthase, such as vascular smooth muscle cells, as well as cells such as those of the myocardium that express considerable levels of the constitutive isoform.
iNOS exhibits negligible activity under basal conditions, but in response to factors such as lipopolysaccharide and certain cytokines, expression occurs over a period of hours. The induced form of the enzyme produces much greater amounts of NO than the constitutive form, and induced NOS appears to be the xe2x80x9cpathophysiologicalxe2x80x9d form of the enzyme because high concentrations of NO produced by iNOS can be toxic to cells. Induction of iNOS can be inhibited-by glucocorticoids and some cytokines. Relatively little is known about postranscriptional regulation of iNOS. Cytotoxic effects of NO are probably largely independent of guanylate cyclase and cyclic GMP formation.
It is known that administration of drugs consisting of nitric oxide, or releasing nitric oxide, can inhibit restenosis after angioplasty. Chronic inhalation of nitric oxide inhibits restenosis following balloon-induced vascular injury of the rat carotid artery. Oral administration of NO donors (drugs which release nitric oxide) inhibits restenosis in rat and pig models of balloon angioplasty-induced vascular injury.
The long term benefit of coronary balloon angioplasty and atherectomy is limited by the considerably high occurrence of symptomatic restenosis (40-50%) 3 to 6 months following the procedure. Restenosis is in part due to myointimal hyperplasia, a process that narrows the vessel lumen and which is characterized by vascular smooth muscle cell migration and proliferation. Medical therapies to prevent restenosis have been uniformly unsuccessful. Intravascular stents have been successfully used to achieve optimal lumen gain, and to prevent significant remodeling. However, intimal thickening still plays a significant role in stent restenosis.
The vascular architecture is maintained or remodeled in response to the changes in the balance of paracrine factors. One of the substances that participates in vascular homeostasis is endothelium derived nitric oxide (NO). NO is synthesized from the amino acid L-arginine by NO synthase. NO relaxes vascular smooth muscle and inhibits its proliferation. In addition, NO inhibits the interaction of circulating blood elements with the vessel wall. NO activity is reduced in hypercholesterolemia and after vascular injury. The administration of L-arginine alone has been shown to restore vascular NO activity in animals and in humans with endothelial vasodilator dysfunction.
We have developed an approach to diminish the incidence of restenosis resulting from angioplasty and atherectomy, using an arginine based mixture to enhance NO activity in the vessel wall.
The term xe2x80x9csubjectxe2x80x9d as used herein means any mammal, including humans, where nitric oxide (xe2x80x9cNOxe2x80x9d) formation from arginine occurs. The methods described herein contemplate prophylactic use as well as curative use in therapy of an existing condition.
The term xe2x80x9cnative NOxe2x80x9d as used herein refers to nitric oxide that is produced through the bio-transformation of L-arginine or in the L-arginine dependent pathway. xe2x80x9cEDRFxe2x80x9d or xe2x80x9cEDNOxe2x80x9d may be used interchangeably with xe2x80x9cnative NOxe2x80x9d. The term xe2x80x9cendpointsxe2x80x9d as used herein refers to clinical events encountered in the course of treating cardiovascular disease, up to and including death (mortality).
xe2x80x9cL-argininexe2x80x9d as used herein includes all biochemical equivalents (i.e., salts, precursors, and its basic form). L-lysine may be considered a biological equivalent of L-arginine. Other bioequivalents of L-arginine may be arginase inhibitors, citrulline, ornithine, and hydralazine.
xe2x80x9cTo mixxe2x80x9d, xe2x80x9cmixingxe2x80x9d, or xe2x80x9cmixture(s)xe2x80x9d as used herein means mixing a substrate (i.e., L-arginine) and another therapeutic agent agonist (e.g., nitroglycerin or an Hmg-CoA reductase inhibitor): 1) prior to administration (xe2x80x9cin vitro mixingxe2x80x9d); 2) mixing by simultaneous and/or consecutive, but separate (e.g., separate intravenous lines) administration of substrate (L-arginine and agonist to cause xe2x80x9cin vivo mixingxe2x80x9d; and 3) the administration of a NOS agonist after saturation with a NOS substrate (e.g., L-arginine is administered to build up a supply in the body prior to administering the NOS agonist (nitroglycerin or Hmg-CoA reductase)); or any combination of the above which results in pre-determined amounts of a NOS agonist and a NOS substrate.
xe2x80x9cAgonistxe2x80x9d refers to an agent which stimulates the bio-transformation of a NO precursor, such as L-arginine or L-lysine to EDNO or EDRF either through enzymatic activation, regulation or increasing gene expression (i.e., increased protein levels of c-NOS). Of course, either or both of these mechanisms may be acting simultaneously.
As used herein, the term xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the hosts to which it is administered.
Methods and devices are provided for inhibiting the pathology associated with vascular injury, particularly during angioplasty and atherectomy. A NO producing mixture, preferably L-arginine and a NOS agonist, is introduced either intraluminally or more preferably intramurally (for example by a stent) to into the walls of the injured vessel in proximity to the injury in an amount to inhibit the pathology, e.g, restenosis, associated with the vascular injury. Various conventional delivery devices may be used for the delivery of the therapeutic mixture.
The following examples or embodiments are offered by illustration, and not by way of limitation.
The present invention is generally directed to treating vessels with a mixture of L-arginine and an agent which enhances the biotransformation of L-arginine into NO. The incidents associated with restenosis are expected to substantially reduced and prevented providing for a reduced incidence of restenosis.
In an embodiment of the present invention there is provided a method for reducing the probability of restenosis. The method comprises introducing intramurally or intraluminally to a site of an injury at a pre-determined time from said injury to a second pre-determined time (e.g. not later than 6 hours thereafter), a therapeutic mixture, said therapeutic mixture including a biological equivalent of L-arginine; and an agent which enhances NO availability. In this embodiment it is preferable that the agent stimulates conversion of L-arginine to NO by nitric oxide synthase, even more preferably the agent is a NOS agonist, even more preferably, the agent is a nitrate, and even more preferably the agent is nitroglycerin. In this embodiment, it is preferable that the biological equivalent of L-arginine be selected from the group consisting of L-arginine and L-lysine. Alternatively, the biological equivalent of L-arginine is selected from the group consisting of citrulline and arginase inhibitors. The agent may also prevent degradation of NO. In this case it is preferable that the agent include DOX.
In an alternative embodiment the present invention provides, a method for reducing the probability of restenosis resulting from vascular injury, comprising: introducing intramurally and preferably proximally to the site of said injury over a predetermined time (preferably about 2 min to 0.5 h) an active agent, wherein the active agent includes a nitric oxide precursor and an Hmg-CoA reductase inhibitor. In this embodiment the nitric oxide precursor is preferably a biological equivalent of L-arginine, even more preferably a biological equivalent is selected from the group consisting of L-arginine or L-lysine or a combination of the two.
An alternative embodiment of the present invention provides a method for reducing the severity of restenosis, comprising introducing a biological equivalent of L-arginine intramurally and proximally to the site of said injury at a time from the time of said injury to a time not later than 6 hours (in an aqueous solution at a concentration in the range of 20 to 500 g/l); and introducing an agent which enhances the conversion of said biological equivalent of L-arginine into nitric oxide. It is preferable that the method further includes the step of introducing an agent which prevents the degradation of said nitric oxide. In this embodiment the step introducing may be by means of a local delivery catheter.
In an alternative embodiment there is provided a method for reducing the probability of restenosis resulting from injury caused by angioplasty or atherectomy, comprising: introducing intramurally or intraluminally, preferably intramurally at the site of said injury a stent, said stent having a body comprised of L-arginine and a NOS agonist.
An alternative embodiment of the present invention provides a stent having a body comprising a NO precursor agent and a NOS agonist, the NO precursor includes at least one of L-arginine or L-lysine, and the a NO precursor agent and NOS agonist releasable under conditions present in a blood vessel.
An alternative embodiment of the present invention provides a stent having a body comprised of L-arginine and a nitrate, preferably nitroglycerin.
An alternative embodiment of the present invention provides a stent having a body comprised of L-arginine and an Hmg-CoA reductase inhibitor, preferably atorvastatin or pravastatin.
An alternative embodiment of the present invention provides a stent having a body comprised of L-arginine and an angiogenic growth factor.
An alternative embodiment of the present invention provides a stent having a body comprised of L-arginine and DOX.
An alternative embodiment of the present invention provides an antirestenosis device comprised of a body, said body including a therapeutic formulation. The therapeutic formulation of this embodiment includes a NO precursor and a NO producing catalytic agent. In this embodiment it is preferable that the NO precursor is L-arginine. Alternatively, the NO precursor may be L-lysine or a combination of L-arginine and L-lysine. In this embodiment the NO precursor may be an arginase inhibitor. In this embodiment the NO producing catalytic agent is preferably a nitrate, preferably nitroglycerin. In this embodiment the NO producing catalytic agent may also be an Hmg-CoA reductase inhibitor, preferably statin, and more preferably, pravastatin. In this embodiment the NO producing catalytic agent may be an angiogenic growth factor. In this embodiment the NO producing catalytic agent may be DOX.
An alternative embodiment of the present invention provides a stent comprised of a body, said body including an arginine based mixture, said arginine based mixture including a biological equivalent of arginine and an agent which enhances the bioavailability of nitric oxide. In this embodiment of the present invention, the biological equivalent of arginine is L-arginine. Alternatively, the biological equivalent of arginine may be L-lysine. In this embodiment the biological equivalent of arginine may be an arginase inhibitor. In this embodiment the agent which enhances the bioavailability of nitric oxide is preferably a nitrate. In this embodiment the agent may be nitroglycerin. In this embodiment the agent may be an Hmg-CoA reductase inhibitor. In this embodiment the agent may a statin, preferably pravastatin. The mixture may also include an angiogenic growth factor or DOX.
Finally as an alternative embodiment of the present invention, there is provided a local in-dwelling intra-arterial eluting drug delivery device comprised of a body, said body incorporating a therapeutic mixture therein, said therapeutic mixture including a NO precursor agent and an agent which enhances the conversion of the precursor agent to native NO. In this embodiment it is preferable that the NO precursor agent is L-arginine. In this embodiment the NO precursor may be L-lysine. In this embodiment the NO precursor agent may be an arginase inhibitor. In this embodiment the agent which enhances the conversion of the precursor agent to native NO may be a nitrate. In this embodiment the agent which enhances the conversion of the precursor agent to native NO may be nitroglycerin. In this embodiment the agent which enhances the conversion of the precursor agent to native NO may be an Hmg-CoA reductase inhibitor. In this embodiment the agent which enhances the conversion of the precursor agent to native NO may be a statin. In this embodiment the agent which enhances the conversion of the precursor agent to native NO may be pravastatin. In this embodiment the agent which enhances the conversion of the precursor agent to native NO may be an antiogenic growth factor. In this embodiment the agent which enhances the conversion of the precursor agent to native NO maybe DOX.